Lipids as a class of molecules display a wide diversity in both structure and biological function. The propensity of hydrophobic lipid tails to self-associate and the tendency of the hydrophilic moieties to interact with aqueous environments and with each other, is the physical basis of the spontaneous formation of the lipid bilayer membrane. The membrane is the supporting matrix for a wide spectrum of proteins involved in many cellular processes. Lipids also allow particular proteins in membranes to aggregate, and others to disperse. Approximately 20-35% of all proteins are integral membrane proteins, and probably half of the remaining proteins function at or near a membrane surface. Lipids can also act as first and second messengers in signal transduction and molecular recognition processes.
Discoidal lipid particles are hypothesized to be intermediates in a micelle-to-vesicle transition reaction of lipids upon detergent removal, as deduced from static and dynamic light scattering experiments (Leng et al. Biophys. J. 85, 1624-1646 (2003)). When a detergent is removed from a mixed-micellar state of lipids and detergents, disc-like intermediate micelles rapidly form. These disc-like micelles grow by coalescence. Large discs then become unstable due to incomplete coverage of their perimeter by detergent which increases the energy per unit length of boundary (line tension) of the disc, leading to closure of the large lipid disc to form a lipid vesicle.
Apolipoproteins are uniquely flexible proteins characterized by a series of proline-punctuated amphipathic, α-helical domains capable of associating with lipid acyl chains. Apolipoproteins have been classified as “protein detergents” capable of isolating membrane proteins in a soluble and planar lipid environment. The formation of discoidal structures of lipids surrounded by human apolipoprotein A-I (apo A-I) was first demonstrated in 1980 (Jonas and Drengler J. Biol. Chem. 255, 2190-2194 (1980)), after similar structures were implicated in transport of cholesterol by High Density Lipoproteins (HDL) in the serum (Jonas et al. Federation Proceedings 36, 829-829 (1977)). An engineered N-terminal truncated form of human apo A-I was used in a similar manner to incorporate peripheral and integral membrane proteins into such bilayer nano discs (Bayburt et al. Nano Lett. 2, 853-856 (2002); Bayburt and Sligar Proc. Natl. Acad. Sci. USA 99, 6725-6730 (2002); Bayburt and Sligar Protein Sci. 12, 2476-2481 (2003)). The presence of apo A-I at a certain concentration possibly replaces the surfactant stabilizing the edge of the bilayer due to lipid-binding properties of its amino acids to provide kinetic stability to the disc-like micelle formed.
Incorporation of peripheral and integral membrane proteins into nanoscale discoidal lipoproteins or nanoscale apolipoprotein bound phospholipid bilayers (NABB) have several advantages over incorporation of those proteins into lipid vesicles. The discrete nanoscale lipid discs prevent aggregation phenomena as seen with detergent micelles and light-scattering of lipid vesicles, thus providing a system that can be isolated in a monodisperse form suitable for multi-well assays.
Another problem is that vesicles form closed structures and the chemical composition of the lumen is difficult to alter. Lipid vesicles are also prone to light scattering artifacts in optical spectroscopy and a precise control of the number of receptors per vesicle, and heterogeneity of vesicle sizes, are difficulties. A further complication, when membrane proteins such as a GPCR are reconstituted in a vesicle, arises from the ‘sidedness’ of the membrane protein interactions with ligands and G proteins respectively.
Chemokine receptors are members of family A GPCRs and of significant clinical importance. Chemokines (chemotactic cytokines) constitute a superfamily of structurally related small proteins, less than 100 residues in length that are implicated in the control of a large variety of biological processes including inflammation, immunosurveillance, viral infection and cancer (Gerard and Rollins Nat. Immunology 2, 108-115 (2001)). CCR5 is an important co-receptor that is exploited for entry of HIV-1 (Human Immunodeficiency Virus type 1) and is critical for the transmission of this virus in the body (Berger et al. Ann. Rev. Immunol. 17, 657-700 (1999)). No adverse health effects have been linked to the non-functional CCR5 in these individuals, which makes CCR5 a very attractive target for pharmaceutical intervention for the prevention of HIV/AIDS (O'Brien and Moore Immunol. Reviews 177, 99-111 (2000)).
A CCR5 to CXCR4 switch has also been reported to accompany the onset of AIDS (Berger et al. Ann. Rev. Immunol. 17, 657-700 (1999)). SDF-1 (Stromal cell-Derived Factor-1) is a CXC-chemokine and is expressed as two functionally identical, alternative splice variants: SDF-1α and SDF-1β (Rossi and Zlotnik Ann. Rev. Immunol. 18, 217-243 (2000)), and binds to CXCR4. CXCR4 is expressed in hematopoietic cell-lines (Zou et al. Nature 393, 595-599 (1998)) and is thought to be involved in signaling responsible in part for stem-cell maturation, naive T-cell migration to spleen for antigen presentation and other developmental signaling (Proudfoot Nat. Rev. Immunology 2, 106-115 (2002)). The SDF-1/CXCR4 system has been implicated in breast and ovarian cancers (Muller Biochem. Pharmacol. 72, 739-748 (2001); Scotton et al. Cancer Res. 62, 5930-5938 (2002)) and brain tumors (Rubin et al. Proc. Natl. Acad. Sci. USA 100, 13513-13518 (2003)).
Chemokine receptors thus provide targets for generating effective antibody responses. Ideally, intact receptors or variants must be presented in a correctly folded, homogeneous, and soluble form at a high concentration capable of eliciting an immune response. Moreover, vaccine candidates must conform to certain levels of purity and defined composition.
Various methods of generating nanoscale apolipoprotein bound phospholipid bilayer (NABB) with associated membrane proteins have been described. Kudlicki et al., disclose in vitro protein synthesis systems where the membrane protein is synthesized in the presence of the NABB (US Patent Application Publication 2007/0117179). However, it is not evident that the in vitro synthesis system can provide for homogeneous populations of NABB with defined lipid and associated protein content or that the associated protein will retain it's native conformation and/or activity. Sligar et al., disclose an in vitro assembly method where the NABBs associated with membrane proteins are obtained after a lengthy dialysis step that provides for removal of solubilizing detergents and subsequent self-assembly of the NABB with associated membrane proteins (U.S. Pat. Nos. 7,083,958 and 7,048,949; US Patent Application Publication US 2005/0182243). However, the lengthy dialysis step used in this in vitro assembly method is inconvenient and may disrupt the native conformation and/or activity of the associated membrane protein.
Accordingly, a need exists for new methods of forming nanoscale apolipoprotein bound phospholipid bilayers (NABBs) that provide for homogenous populations of NABBs with defined lipid and protein compositions. New methods for associating membrane proteins into NABB that provide for increased activity and/or native conformations of the integrated proteins are also needed, as are the resultant NABB compositions comprising integrated membrane proteins with increased activity and/or native protein conformations.